Motor proteins, or mechanoenzymes, convert metabolic energy directly into displacement, powering motion at the subcellular level in most living organisms. The largest class of motor proteins is fueled by adenosine triphosphate (ATP) and includes members of the myosin, dynein, and kinesin "superfamilies" of proteins. Despite more than a century of study and an arsenal of approaches, the mechanisms by which these motor proteins function are not firmly established. The mystery of motility remains one of the outstanding problems in biology, and it bears a direct relationship to understanding the molecular basis of motor-related human disease. Members of the kinesin motor superfamily have been implicated in a long list of important ailments, including diabetes, Alzheimer's disease, hereditary spastic paraplesia, Charcot-Marie-Tooth disease, Kartagener's syndrome, polycystic kidney disease, and motor neuron disease. With the advent of specialized techniques, particularly those in the new field of single molecule biophysics, we are tantalizingly close to achieving an understanding of kinesin motor mechanism. Among all motor proteins, members of the kinesin superfamily offer special advantages for research, because (1) they represent the smallest - and arguably the simplest - motors known; (2) processive (continuous) motion is generated by individual motors, facilitating experimental study; (3) atomic-level structural information is available; (4) functional, recombinant forms of kinesin can be expressed in bacteria or various cell lines; and (5) new technology exists that can supply precisely-controlled loads and measure nanometer-level displacements for individual molecules. My lab has played a major role in the development of much of this new technology, particularly laser-based optical traps ("optical tweezers") and single-molecule fluorescence approaches. Single-molecule methods have already led to breakthroughs in our understanding. The long-term goal of my research is to develop a quantitative understanding of how kinesin proteins work, based on single-molecule physiology combined with biochemical and biostructural information. Specific aims of this grant include detailed measurements of the speeds, forces, displacements, ATP coupling, head-head interactions, and other properties of kinesin motors at the single-molecule level. For this next phase of research, we plan to study not only conventional kinesin (kinesin-1, an intracellular vesicle transporter), but also carry out parallel studies of unconventional members of the kinesin superfamily, such as Eg5 (kinesin-5) and KIF3A/B (kinesin-2), two motors known to play key roles in cell division (mitosis), and which therefore represent targets for novel anti-cancer drugs, several of which are now in clinical trials. [unreadable] [unreadable] [unreadable]